Gram-negative bacteria are the causative agents of a variety of important human diseases. Successful treatment of infections with these organisms is hampered by the complex cell envelope that surrounds them. This envelope includes a second (outer) membrane layer that is difficult for drugs to penetrate. Gram-negative bacteria thus have a high intrinsic resistance to antibiotics and are completely insensitive to many drugs that are effective against bacteria lacking an outer membrane. In this project, we intend to validate new targets for antibiotics that when inactivated will compromise the permeability barrier of the outer membrane to either kill gram-negative bacteria or eliminate their intrinsic resistance to approved therapeutics. The project is based on our studies of cell division in Escherichia coli. In this model gram-negative bacterium, we recently identified factors required for cell envelope remodeling during division and have developed tools to study them. During division, new cell wall material produced by the cytokinetic machinery must be processed by hydrolytic enzymes for daughter cells to separate. We have discovered that this processing is carried out by enzymes called amidases that are activated by factors with LytM domains. Once the cell wall is processed, a second set of factors called the Tol-Pal system catalyzes the constriction of the outer membrane to complete the division process. Inactivation of either the amidases or the Tol-Pal system in E. coli and other gram-negative bacteria leads to the formation of long chains of cells that cannot separate. These cell chains have been shown to have a compromised outer membrane permeability barrier and are hypersensitive to many drugs. Despite their role in maintaining the outer membrane permeability barrier, the importance of these cell separation systems for drug resistance has not been investigated in problematic gram-negative pathogens. Therefore, in this project, we will validate cell separation systems as potential drug targets in te pathogen Pseudomonas aeruginosa. Molecular genetics will be used to investigate the effect of inactivating these systems on the growth and drug resistance of this organism, and chemical screens will be implemented to identify inhibitors of the process. Although we focus on P. aeruginosa, the systems chosen for targeting are highly conserved in gram-negative bacteria. Thus, our results will be broadly relevant to the development of novel treatments for gram-negative infections.